rDNA介导的乳酸克鲁维酵母整合型表达载体的构建及初步验证  

Construction and Verification of a Ribosomal DNA-Mediated Integrative Expression Vector in Kluyveromyces lactis

作　　者：孙海烨 张梁 李由然[1] 顾正华 丁重阳 石贵阳 SUN Haiye;ZHANG Liang;LI Youran;GU Zhenghua;DING Zhongyang;SHI Guiyang(National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China;Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China)

机构地区：[1]粮食发酵工艺与技术国家工程实验室,江南大学,江苏无锡214122 [2]江南大学工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《食品与生物技术学报》2019年第10期87-97,共11页Journal of Food Science and Biotechnology

基　　金：国家863计划项目(2011AA100905);江苏省杰出青年科学基金项目(BK20140002);国家星火计划重点项目(2015GA690004)

摘　　要：构建一个适用于乳酸克鲁维酵母(Kluyveromyces lactis)稳定的整合型表达载体,并通过鼠灰链霉菌(Streptomyces murinus)来源的腺苷酸脱氨酶基因(AMPD)的表达对该载体进行初步验证。以质粒pMD 19T-simple为空骨架,K.lactis GG799来源的18S rDNA序列为同源重组位点,酿酒酵母(Saccharomyces cerevisiae)来源的半乳糖苷酶启动子(pScGAL7)为外源基因调控元件,乙酰胺酶基因(amdS)为筛选标记,AMPD为报告基因,构建重组表达载体pTRGA-amdS。载体经Sac II线性化后,去除了大肠杆菌(Escherichia coli)来源的ori和氨苄青霉素抗性基因(Ampr),将同源重组片段电转化K.lactis GG799得到重组菌,利用实时荧光定量PCR(RT-QPCR)测定重组菌AMPD基因整合拷贝数为1~3,AMP脱氨酶酶活测定结果表明:酶活与拷贝数呈正相关,含3个整合拷贝的重组菌在半乳糖诱导下酶活提高了32.6%,达到(590±13.33)U/mL。在无选择压力下重组菌连续生长58世代稳定性为98.59%。通过载体pTRGA-amdS的构建与应用实现了外源基因在K.lactis中的稳定表达,初步探究了18S rDNA在同源重组中的作用,为K.lactis的进一步分子改造提供参考。An integrative expression vector applied for Kluyveromyces lactis was constructed in this study.To verify its application,the adenylate deaminase gene(AMPD)derived from Streptomyces murinus was sucessfully expressed by this vector.The recombinant vector pTRGA-amdS that based on the plasmid pMD 19T-simple was established containing K.lactis-derived 18S rDNA sequence for homologous recombination,the Saccharomyces cerevisiae-derived galactosidase gene promoter(pScGAL7)for expression of heterologous genes,the acetamidase gene(amdS)as selection marker,and AMPD as reporter gene.The recombinant expression vector was linearized by Sac II,removing E.coli ori and Amp resistant gene,then electransformed into K.lactis GG799 to obtain transformants.Real-time quantitative PCR analysis indicated that the copy number of the integrated AMPD gene ranged from 1 to 3 and positively correlated with AMP deaminase activity.When the inducer galactose was used as carbon source,the AMP deaminase activity in the culture supernant of the recombinant with three AMPD gene copies reached to 590±13.33 U/mL,improved 32.6%.The integrants were mitoically stable for 58 generations under the non-selective pressure condition(98.58%).We constructed a new vector with the stable expression ability of heterologous gene and confirmed the 18S rDNA as a suitable recombination site.These research results lay a foundation for further molecular modification of K.lactis.

关 键 词：乳酸克鲁维酵母 18S rDNA 整合表达 AMP脱氨酶 RT-QPCR 拷贝数 

分 类 号：Q78[生物学—分子生物学]